Unknown,Transcriptomics,Genomics,Proteomics

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Cultures of hiPS cells and their differentiation into neuronal cells

ABSTRACT: In order to identify endogenous factors that modulate Aβ production, we performed a transcriptome analysis on neuronal cells derived from human induced pluripotent stem cells (hiPS) because these cells changed the Aβ production during neuronal differentiation. When the expression of APP and the ratio Aβ42/40 of the secreted proteins were measured at days 38, 45, and 52 during differentiation, both APP expression and the secretion of Aβ40 and Aβ42 at days 45 and 52 were significantly increased compared to those at day 38. Furthermore, the ratio of Aβ42/40 was dramatically reduced at days 45 and 52 compared to that at day 38. We hypothesized that transcriptional changes during the differentiation into neuronal cells may affect APP production and/or processing. To test this hypothesis, we performed microarray analyses using three independent neuronal cell cultures that were differentiated from each of three hiPS cells, namely 201B7, 253G4, and AD4F-1 (nine cell lines in total). Total RNA was prepared at three differentiation time points (days 38, 45, and 52) and subjected to an Affymetrix Gene Chip Human Exon 1.0 ST array. Compare between samples from 38-day cultures and samples from 45- and 52-day cultures.

ORGANISM(S): Homo sapiens

SUBMITTER: Tsuneyoshi Seki

PROVIDER: E-MTAB-7852 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Astrocytes, one of the most resilient cells in the brain, transform into reactive astrocytes in response to toxic proteins such as amyloid beta (Aβ) in Alzheimer’s disease (AD). However, reactive astrocyte-mediated non-cell autonomous neuropathological mechanism is not fully understood yet. We aimed our study to find out whether Aβ-induced proteotoxic stress affects the expression of autophagy genes and the modulation of autophagic flux in astrocytes, and if yes, how Aβ-induced autophagy-associated genes are involved Aβ clearance in astrocytes of animal model of AD. Whole RNA sequencing (RNA-seq) was performed to detect gene expression patterns in Aβ-treated human astrocytes in a time-dependent manner. To verify the role of astrocytic autophagy in an AD mouse model, we developed AAVs expressing shRNAs for MAP1LC3B/LC3B (LC3B) and Sequestosome1 (SQSTM1) based on AAV-R-CREon vector, which is a Cre recombinase-dependent gene-silencing system. Also, the effect of astrocyte-specific overexpression of LC3B on the neuropathology in AD (APP/PS1) mice was determined. Neuropathological alterations of AD mice with astrocytic autophagy dysfunction were observed by confocal microscopy and transmission electron microscope (TEM). Behavioral changes of mice were examined through novel object recognition test (NOR) and novel object place recognition test (NOPR). Here, we show that astrocytes, unlike neurons, undergo plastic changes in autophagic processes to remove Aβ. Aβ transiently induces expression of LC3B gene and turns on a prolonged transcription of SQSTM1 gene. The Aβ-induced astrocytic autophagy accelerates urea cycle and putrescine degradation pathway. Pharmacological inhibition of autophagy exacerbates mitochondrial dysfunction and oxidative stress in astrocytes. Astrocyte-specific knockdown of LC3B and SQSTM1 significantly increases Aβ plaque formation and hypertrophic reactive astrocytes in APP/PS1 mice, along with a significant reduction of neuronal marker and cognitive function. In contrast, astrocyte-specific overexpression of LC3B reduced Ab aggregates in the brain of APP/PS1 mice An increase of LC3B and SQSTM1 protein is found in astrocytes of the hippocampus in AD patients. Taken together, our data indicates that Aβ-induced astrocytic autophagic plasticity is an important cellular event to modulate Aβ clearance and maintain cognitive function in AD mice.

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Cultures of hiPS cells and their differentiation into neuronal cells

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